This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The nicotinic acetylcholine (ACh) receptor (nAChR) is the prototypical agonist-gated ion channel responsible for rapid excitatory neurotransmission. The nAChR agonists include many toxicants, potential therapeutic agents, and the important neonicotinoid insecticides. Initial attempts to understand receptor-agonist interactions involved site-directed or chimeric mutagenesis, estimating the role of specific region(s) or amino acid(s) on the pharmacological response (electrophysiological or ligand binding). The structural biology approach of high-resolution X-ray crystallography reveals geometries of functional amino acids in the drug-bound state and conformational rearrangement of the protein upon ligand interaction. Alternatively, incorporation of an unnatural amino acid or photoaffinity labeling provides a direct and physiologically-relevant definition of the recognition properties of the ligand binding environment. Binding site interactions of nicotinoid and neonicotinoid agonists have been characterized by comparative structural and chemical biology approaches using mollusk ACh binding protein (AChBP), which is a suitable structural surrogate of the extracellular ligand-binding domain of the nAChR. 5-Azido-6-chloropyridin-3-yl nicotinic photoaffinity probes have played an important role in studying agonist-nAChR interactions. In principle, these probes bind to the specific site and then the reactive nitrene intermediate, generated by photoirradiation, reacts covalently with the target protein. On photoaffinity labeling of Aplysia californica AChBP, the azidochloropyridinyl photoprobes derivatized at only one position at the interface between loop C Y195 on the principal or (+)-face subunit and loop E M116 on the partnering or (-)-face subunit, establishing a bound ligand position and conformation identical to those observed in crystal structures. Interestingly, a nicotinic photoaffinity ligand of this type exclusively labeled the [unreadable]4 subunit of the chick [unreadable]4[unreadable]2 nAChR subtype, in which loop C Y225 on the [unreadable]4 subunit and loop E F137 on the [unreadable]2 subunit are spatially equivalent to Y195 and M116, respectively, of AChBP. These observations may be rationalized by the difference in the reactivity between amino acids and the photoactivated probe molecule. Therefore, the present investigation designs a methionine- and tyrosine-scanning approach principally on the loop E domain involving 17 AChBP mutants regarding photoreactivity of the 5-azido-6-chloropyridin-3-yl nicotinic photoprobes, ultimately mapping the specific site undergoing photoderivatization by mass spectrometry analyses and precise position and conformation of the bound ligand.